Field of the Invention
The present invention relates to novel bioink which is biomaterial in the form of water dispersion of cellulose nanofibrils and can be converted into desired 3D shapes using 3D Bioprinting technology.
This novel bioink is suitable for 3D cell culturing and growing living tissues and organs. In this invention, cellulose nanofibrillar material is processed through different mechanical, enzymatic and/or chemical steps to yield fibril dispersion with desired rheological and morphological properties to be used as bioink in a 3D Bioprinter. The homogenization processes can be followed by purification of the material to yield biomaterial which has a desired level of cytocompatibility and can thus be combined with living cells.
Cellulose nanofibrils can be produced by a microbial process but can also be isolated from plant's secondary or primary cell wall, animals such as tunicates, algae and fungi. The desired parameters described in this invention are the size of fibril, the surface properties, concentration and biocompatibility. In this invention cellulose nanofibrils are combined with different additives which facilitate a crosslinking process to enhance mechanical properties of 3D Bioprinted structures. The nanocellulose bioink, CELLINK™, is typically prepared using sterile components and prepared in clean room conditions. The osmolarity of the CELLINK™ is designed to provide compatibility with mammalian cells. CELLINK™ can be 3D Bioprinted with cells or without cells. CELLINK™ can also be used to support other bioinks such as materials prepared from decellularized tissue and organs.
More particularly, embodiments of the invention relate to soft and hard tissue repair, scaffolds, systems and methods for the design, production, and control of the architecture and biomechanical properties of biomaterials which are used to grow tissue and organs. Specific embodiments of the invention relate to biocompatible materials, tissue engineering and regenerative medicine, implants, biomedical devices and health care products and, more particularly, to use as bioink in 3D Bioprinting processes to create optimal architecture and biomechanical performance of artificial tissues and organs. The present invention also relates to novel devices, systems and methods employing engineered tissues and/or organs having a desired 3D architecture and morphology supported by a 3D nanocellulose based scaffold, which can be used for high throughput drug discovery, screening, and toxicity testing. It can also be used to grow artificial tumor and thus used for in vitro cancer research.
Description of Related Art
Tissue engineering is using cells, supporting material—scaffolds, growth factors and in many cases bioreactors, to grow in vitro or in vivo tissue and organs. The driving force has been a shortage of organs which are needed for transplantation. Tremendous scientific and technological progress has been made in the past 20 years which has made it possible to grow almost all human tissues and many organs. In recent years the pharmaceutical and cosmetic industry has shown great interest in applying advances in tissue engineering to grow tissue and “mini” organs for drug discovery and drug testing. The new regulations are making restrictions for using animals for testing of cosmetic products. This has initiated tremendous interest for developing human skin models “skin on the plate”.
The human cells should have a 3D environment similar to a native tissue environment to be able to migrate, proliferate, and/or differentiate to develop functional tissues. Likewise, stem cells typically need a 3D environment to differentiate into desired cell lineage. This is the reason why scaffolds with 3D architecture and specific microporosity have been developed for tissue engineering applications. In classical tissue engineering experiments, cells are seeded in a 3D scaffold and then cultivated in an incubator or stimulated in a bioreactor or directly implanted in vivo.
Many different synthetic and natural polymers have been evaluated as scaffolds for tissue engineering. Examples of biodegradable synthetic polymers include polylactic and polyglycolic acid. These polymers have often fast degradation characteristics and/or produce an environment which causes inflammation. Natural polymers include collagen, hyaluronic acid and its derivatives, alginate, and chitosan. While these materials can be fabricated into films, meshes, or more complex 3D structures, their successful use is limited by their physical and biochemical properties. Fabrication of 3D structures with controlled architecture and interconnected porosity has been challenging. The methods used, such as freeze drying, porogen removal or electrospinning, show poor reproducibility and lack of control of 3D architecture in micro scale. As the consequence of that, there have been difficulties in cell seeding since cell migration requires good pore interconnectivity.
In 3D printing processes, an object is fabricated layer by layer by a printer device using computer aided design, CAD file. 3D printing has been already successfully used in tissue engineering by many scientists to fabricate patient specific scaffolds. The scaffolds made of thermoplastic polymers have been extruded using 3D printers. The disadvantage of 3D printing using thermoplastic materials is a difficulty in cell seeding due to limited cell migration into porous structures. 3D Bioprinting operates using liquids in room or body temperature and thus can potentially handle living cells. The introduction of 3D Bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine, which might enable the reconstruction of living tissue and organs preferably using the patient's own cells. The 3D bioprinter is a robotic arm able to move in the X,Y,Z directions with a resolution of 10 μm while dispensing fluids. The 3D bioprinter can position several cell types and thus reconstruct the architecture of complex organs.
In U.S. Pat. No. 8,691,974 B2, entitled “Three-dimensional Bioprinting of Biosynthetic Cellulose Scaffolds for Tissue Engineering,” a novel fermentation technique for controlling 3D shape, thickness and architecture of the entangled cellulose nanofibril network was presented. That patent described the use of a fermentation process to grow a 3D structure of biosynthetic cellulose. This technique can unfortunately not be combined with mammalian cells due to the differences in cultivation conditions at 37 degrees, which is required for mammalian cells, since bacterial cells are killed. Biosynthetic cellulose, BC is an emerging biomaterial for biomedical devices and implants (Petersen N, Gatenholm, P., Bacterial cellulose-based materials and medical devices: current state and perspectives, Applied Microbiology and Biotechnology, 91, 1277, 2011). The BC nanofibrils have a similar size and morphology as collagen (diameter 10-30 nm and length up to micrometers), which is very attractive for cell attachment, cell migration, and the production of Extracellular Matrix components. In vitro and in vivo studies have shown that BC implants typically do not elicit any foreign-body reaction, fibrosis, and/or capsule formation, and/or connective tissue integrates well with BC biomaterial (Helenius G, H. Bäckdahl, A. Bodin, U. Nanmark, P. Gatenholm, B. Risberg, In vivo Biocompatibility of Bacterial Cellulose, J. Biomed. Mater. Res. A., 76, 431, 2006; Martinez Avila, H., S. Schwarz, E. M. Feldmann, A. Mantas, A. Von Bomhard, P. Gatenholm, and N. Rotter, Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration. Appl. Microbiol. Biotechnol., 2014. 98(17): p. 7423-7435.).
It is expected that a biosynthetic cellulose network cannot as such be used as a scaffold for tissue engineering because the relatively tight network of cellulose nanofibrils which make cell migration difficult to impossible. The biofabrication processes in which the macroporosity of 3D nanocellulose biomaterial has been developed by introducing porogens during the fermentation process has been described Bäckdahl, H., Esguerra, M., Delbro, D., Risberg, B., and Gatenholm, P., Engineering microporosity in bacterial cellulose scaffolds, Journal of Tissue Engineering and Regenerative Medicine, 2 (6), 320-330 (2008). The porogens have to be removed during purification process. None of the methods enable reproducible and scalable control of the architecture of the scaffolds or a convenient method to combine with the cells.
The development of high resolution 3D Bioprinters enables positioning of several human cell types with high accuracy and reproducibility and thus reconstruction of complex tissue and organs. Rapid advances in stem cell isolation from patient tissue, such as adipose, make it possible to have access to a sufficient amount of autologous cells for tissue repair in one step surgery. The cells typically cannot be printed alone since they are expected not to stay in place. As a result, the cells are suspended in culture medium or buffer, which has a low viscosity. In addition, the cells are preferably protected from high shear stresses in the printing head device. Furthermore, after printing, the cells should be in a cytocompatible environment, which will allow nutrients and oxygen to be administrated to cells and preferentially provide support for cell attachment. When tissue with a desired 3D architecture on different length scales is desired, there is a need of a bioink capable of providing viscoelastic characteristic to be transferred in 3D scaffolds with predetermined shape. The bioinks are preferred to be developed and commercialized to secure a supply of printable and cell friendly scaffolds for tissue engineering and regenerative medicine applications.